EP3947458A1 - Polypeptides de liaison anti-alpha bêta tcr à fragmentation réduite - Google Patents

Polypeptides de liaison anti-alpha bêta tcr à fragmentation réduite

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Publication number
EP3947458A1
EP3947458A1 EP20721354.7A EP20721354A EP3947458A1 EP 3947458 A1 EP3947458 A1 EP 3947458A1 EP 20721354 A EP20721354 A EP 20721354A EP 3947458 A1 EP3947458 A1 EP 3947458A1
Authority
EP
European Patent Office
Prior art keywords
binding polypeptide
antibody
cell
amino acid
disease
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP20721354.7A
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German (de)
English (en)
Inventor
Huawei Qiu
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Genzyme Corp
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Genzyme Corp
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Filing date
Publication date
Application filed by Genzyme Corp filed Critical Genzyme Corp
Publication of EP3947458A1 publication Critical patent/EP3947458A1/fr
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • Monoclonal antibodies are an important class of binding polypeptides and biologic therapeutic drugs.
  • polypeptide backbones are highly stable under physiological conditions. Nevertheless, fragmentation of heavy and light chain polypeptides is a major concern with therapeutic monoclonal antibodies.
  • protein backbones are highly stable under physiological
  • fragmentation may be caused by a variety of mechanisms, e.g., due to the disruption of native covalent bonds, resulting in the cleavage of polypeptide backbones through spontaneous or enzymatic reactions.
  • specific regions and motifs e.g., the Asn-Pro motif
  • Fragmentation may occur at any step during the manufacture or storage of biologic compositions. Because fragmentation may result in reduced potency or the presence of unwanted and potentially immunogenic species, reducing fragmentation is a key consideration in the production of any biologic therapeutic.
  • the present disclosure provides improved compositions and methods useful for treating T-cell-mediated diseases and disorders.
  • humanized binding polypeptides that specifically bind the alpha beta T-cell receptor (abTCR).
  • the anti- abTCR compositions provided herein are an improvement over known compositions in that the improved compositions comprise at least one amino acid substitution or modification that increases the stability of the binding polypeptide by reducing fragmentation of the light chain variable region.
  • methods for treating T-cell-mediated diseases and disorders e.g , graft-versus-host-disease, autoimmune disease, and graft rejection
  • the methods provided herein generally involve administering to a subject in need thereof an effective amount of a humanized binding polypeptide that specifically binds the abTCR.
  • binding polypeptide that specifically binds to human abTCR/CD3 complex, comprising a heavy chain variable region, a light chain variable region, and a constant region, wherein:
  • the light chain variable region comprises three complementarity determining regions (CDRs) LCDR1 , LCDR2, and LCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 26, 27, and 28, respectively;
  • CDRs complementarity determining regions
  • SEQ ID NO: 28 comprises the amino acid sequence Q-Q-W-S-S- X 1 -X 2 -L-T, wherein X 1 is an amino acid selected from the group consisting of Q, D, H, S, Y, and A, and X 2 is an amino acid selected from the group consisting of P and A; and
  • the constant region is of human origin.
  • X 1 is S.
  • X 2 is P.
  • X 1 is S
  • X 2 is P
  • the light chain variable region further comprises a human light chain framework region set forth in SEQ ID NO: 14.
  • the binding polypeptide has increased stability at pH greater than 5 0 compared to VH31
  • the binding polypeptide has increased stability at a temperature greater than 4°C compared to VH31.
  • the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 7, 12, 13, 15, and 16
  • the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 12, and SEQ ID NO: 13
  • the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 15 and SEQ ID NO: 16.
  • the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 15.
  • the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 16.
  • the constant region comprises an Fc modification with a modified glycosylation pattern that reduces Fcy receptor binding.
  • the Fcy receptor is one or more receptor selected from the group consisting of FcyRIlla and FcyRI.
  • the Fc modification is selected from the group consisting of N297Q/S298N/Y3G0S, S298N/T299A/Y30QS, and S29SN/Y30GS.
  • the Fc modification is N297Q/S298N/Y300S.
  • the Fc modification is S298N/T299A/Y300S. In an embodiment, the Fc modification is S298N/Y3GGS.
  • the binding polypeptide is humanized.
  • the binding polypeptide is a monoclonal antibody.
  • the binding polypeptide is multispecific.
  • the binding polypeptide is bispecific.
  • composition comprising a binding polypeptide described herein, and a pharmaceutically acceptable carrier or diluent.
  • a formulation comprising a pharmaceutical composition described herein.
  • the pharmaceutical composition described herein in another aspect, there is provided a formulation comprising a pharmaceutical composition described herein.
  • formulation is a liquid formulation. In certain embodiments, the formulation is a lyophilized formulation.
  • a method of treating a subject for a T-cell- mediated disease or disorder comprising administering to the subject an effective amount of a binding polypeptide or a pharmaceutical composition described herein, such that treatment is achieved.
  • the T-cell-mediated disease or disorder is selected from the group consisting of systemic lupus
  • erythematosus SLE
  • RA rheumatoid arthritis
  • UC ulcerative colitis
  • CD Crohn's disease
  • MS multiple sclerosis
  • scleroderma type 1 diabetes (T1 D)
  • type 1 diabetes T1 D
  • PV pemphigus vulgaris
  • psoriasis psoriasis
  • atopic dermatitis celiac disease, chronic obstructive lung disease
  • Hashimoto’s thyroiditis Graves' disease (thyroid), Sjogren’s syndrome, Guiliain-Barre syndrome, Goodpasture’s syndrome
  • Addison’s disease Wegener’s granulomatosis, primary biliary sclerosis, sclerosing cholangitis, autoimmune hepatitis, polymyalgia rheumatica, Raynaud’s phenomenon, temporal arteritis, giant cell arteritis, autoimmune hemolytic anemia, pernicious anemia,
  • nucleic acid encoding a binding polypeptide described herein.
  • a vector comprising a nucleic acid described herein.
  • a cell comprising a nucleic acid described herein.
  • the cell is a mammalian cell.
  • the mammalian cell is selected from the group consisting of a Chinese hamster ovary (CHO) cell and a human embryonic kidney (HEK) cell.
  • FIG. 1 is a photograph of a protein gel showing the appearance of low molecular weight (LMW) fragments of reference humanized anti- abTCR antibody VH31 formulated antibody under accelerated conditions involving elevated LMW
  • HC heavy chain
  • LC light chain
  • FIG. 2 is a line graph plotting the potency of the reference VH31 formulated antibody at varying concentrations under accelerated conditions for five weeks.
  • the open squares correspond to the reference VH31 antibody stored at 45°C, pH 8.0; open circles correspond to the reference VH31 antibody stored at 45°C, pH 5.0; and open diamonds correspond to the reference VH31 antibody stored under control conditions, i.e., ⁇ 0°G; pH 5.0.
  • FIG. 3 is a photograph of a protein gel showing assigned identities, based on molecular weight and N-terminal sequencing, of the reference VH31 bands which were present under the indicated accelerated conditions.
  • HC heavy chain
  • LG light chain.
  • FIG. 4 depicts the reference VH31 light chain (LG) amino acid sequence (SEQ ID NO: 25) and the N93/P94 putative dipping site.
  • FIGs. 5A-5C are line graphs showing the various light chain fragments resulting from clipping at N93/P94 of trypsin digestion products (amino acids 61-102 or 61 -106 according to Kabat numbering) of reference VH31 light chain. These fragments correspond to amino acid residues 94-102 (FIG. 5A), residues 61 -93 (FIG. 5B), and residues 94-106 (FIG. 5C) of the VH31 LG. The 102 and 106 C-termini are due to incomplete tryptic digestion.
  • FIG 6 depicts proposed process by which cleavage at Asn 93 (N93) may occur in solution.
  • FIG. 7 depicts the various VH31 LC amino acid mutations that were made to enhance LC stability.
  • FIG. 8 is a pair of photographs of protein gels showing the high expression and purity of VH31 antibodies containing either a mutated (lanes 1 -8) or a non- mutated LC. 1 , N93G; 2, N93D; 3, N93H; 4, N93S; 5, N93Y; 6, N93A; 8, N93D/R94D.
  • FIG. 9 is a photograph of a pair of protein gels (left panel) and three SEC- HPLC plots (right pane!) showing that the purity of an antbody comprising a N93S LC mutation was comparable to wildtype (WT) and control (VH31 ) antibodies.
  • FIG. 10 is a line graph plotting the potency of VH31 antibodies comprising the indicated LC mutations.
  • An antibody comprising LC variant N93S (open diamond symbols) was only slightly less potent than either the reference VH31 (open square symbols) or wildtype (WT) antibody (open octagon symbols).
  • FIG. 11 is a photograph of a protein gel showing (left panel) that the N93S mutant had demonstrably reduced degradation compared to either the wildtype (WT) or the VH31 control antibody upon 6 weeks storage at 45°C, pH 8.
  • the right panel shows that the N93S mutant had markedly less loss of potency under accelerated conditions (3-fold loss) compared to either the wildtype or VH31 control antibodies (each greater than 20-fold loss).
  • FIG. 12 is a photograph of a protein gel showing fragmentation of the reference VH31 antibody and the N93S mutant following storage for six weeks at elevated temperature (45°C) at pH 5.5.
  • the present disclosure provides improved compositions and methods for treating T-cell-mediated disorders (e.g., graft-versus-host-disease, autoimmune disease, and allograft rejection).
  • the methods provided herein generally involve administering to a subject in need thereof an effective amount of a humanized binding polypeptide that is specific to the alpha beta T-cell receptor ( abTCR).
  • abTCR alpha beta T-cell receptor
  • compositions in that the improved compositions comprise one or more one amino acid substitution or modification that improves stability of the binding polypeptide by reducing fragmentation of the light chain variable region.
  • the human abTCR/003 complex is a T-cell receptor complex presented on the surface of T cells. See, Kuhns et al. (2006) Immunity 24:133-139. This complex is targeted by the murine monoclonal antibody BMA031 (see, European patent application EP 0403156; SEG ID NOs: 1 and 2, incorporated herein by reference in its entirety) and the humanized and stabilized antibodies disclosed herein.
  • the mouse IgG2b monoclonal antibody BMA031 (Borst et al. (1990) Hum. Immunol. 29(3): 175-88; EP0403156) is specific for the common determinant on the alpha-beta TCR/CD3 complex, and does not bind to the gamma-delta TCR.
  • BMAQ31 is highly immunosuppressive and is capable of inducing apoptosis of activated T cells via a mechanism of activation-induced cell death (AICD) (Wesselborg et al. (1993) J. Immunol. 150(10): 4338-4345).
  • BMA031 does not engage human Fc gamma receptors (FcyR) in the majority of the human population. As such BMA031 does not cause T-cell activation via cross- linking of the T-cell receptor and, therefore, it does not induce T-cell activation or the associated cytokine release. In this regard its profile is highly preferable over that of OKT3.
  • BMD031 is a murine antibody and, as such, is not suitable for repeat dosing in human subjects in view of the human anti-mouse antibody (HAMA) response elicited therein.
  • EUCIV3 successfully bound to T cells; however, as noted in EP 0403156, binding to the abTCR is not as effective as the parent BMAQ31 antibody as determined by flow cytometry competition assays.
  • EUCIV3 was originally generated on a wild-type human lgG1 or lgG4 backbone which still retains FcgR binding. These humanized antibodies therefore allowed for T-cell activation, proliferation and the concomitant cytokine release and as such were significantly different from the original properties of BMA031
  • binding protein or“binding polypeptide,” unless indicated otherwise, is used to refer to a polypeptide (e.g., an antibody or antigen-binding fragment thereof) that contains at least one binding site which is responsible for selectively binding to a target antigen of interest (e.g., a human antigen).
  • exemplary binding sites include an antibody variable domain, a ligand binding site of a receptor, or a receptor binding site of a ligand.
  • the binding polypeptides described herein comprise multiple (e.g., two, three, four, or more) binding sites.
  • antibody unless indicated otherwise, is used to refer to entire antibodies as well as antigen-binding fragments of such antibodies.
  • the term encompasses four-chain lgG molecules, as well as antibody fragments.
  • antibody fragment refers to portions of an intact full-length antibody, for example, as further described below.
  • Antibodies may be of any class, such as IgG, IgA, IgE, IgD, or IgM; and of any subclass, such as IgG1 or lgG4.
  • Different classes and subclasses of immunoglobulin have different properties, which may be advantageous in different applications.
  • lgG4 antibodies have reduced binding to Fc receptors.
  • Naturally occurring immunoglobulins have a common core structure in which two identical light chains (about 24 kDa) and two identical heavy chains (about 55 or 70 kDa) form a tetramer.
  • the amino-terminal portion of each chain is known as the variable (V) region and can be distinguished from the more conserved constant (C) regions of the remainder of each chain.
  • CDRs complementarity determining regions
  • CDR and FR regions and a numbering system have been defined by Kabat et al. (Kabat, E.D. et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, U.S Government Printing Office (1991 ), and updates thereof which may be found online).
  • CDR region boundaries have been further defined by IMGT nomenclature.
  • A“humanized monoclonal antibody,” as used herein, is an antibody which is composed of a human antibody framework, into which have been grafted
  • CDRs complementarity determining regions
  • Changes in the human acceptor framework may also be made.
  • Procedures for the design and production of humanized antibodies are well known in the art, and have been described, for example, in U.S. Patent No. 4,816,397; U.S. Patent No. 4,816,567; and U.S. Patent No. 5,225,539; European Patent Application 0 120 694; European Patent Application 0 125 023; European Patent Application 0 194 276 B1 ; European Patent Application 0 239 400; European Patent Application 0 519 596; and
  • Variable regions of antibodies according to the described embodiments may be obtained, at least in part, by humanizing BMA031 , i.e. , by transferring the CDRs of BMA031 to a human framework, and further modifying one or more VL CDR to improve the stability of the binding polypeptide by reducing fragmentation of the light chain variable region.
  • Two series of humanized BMA031 antibodies are described in PCT Publication WO 2013/037484, which is incorporated by reference herein. These two series are the HEBE1 series, comprising instant SEQ ID NOs: 5-7, 12 and 13, and the GL1 BM series, comprising heavy chain variable regions as shown in instant SEQ ID NOs: 8, 15 and 16.
  • the light chain variable region used is as shown in instant SEQ ID NO: 14 (GL1 BM VK43).
  • the human frameworks used are IGH3-23 (instant SEQ ID NO: 17) in the case of HEBE1 , and IGHV1-3*01 and 1GKV3-11 *01 (instant SEQ ID NOs: 18 and 19, respectively) in the case of GL1 BM.
  • Constant regions may be derived from any human antibody constant regions. Variable region genes may be cloned into expression vectors in frame with constant region genes to express heavy and light immunoglobulin chains. Such expression vectors can be transfected into antibody producing host cells for antibody synthesis.
  • Human antibody variable and constant regions may be derived from
  • immunoglobulin sequences are available in the IMGT/LIGM database (Giudicelli et al. , (2006) Nucleic Acids Res. 34 (suppl. 1 ):
  • A“delta ab” or Aab modification, as used herein, is an Fc modification as described in Armour et al., (1999) Eur. J. Immunol. 29:2613-2624.
  • Techniques for modifying glycosylation of antibody Fc regions are known in the art, and include chemical, enzymatic and/or mutational means, for example, mutation of the N297 position in the CH2 domain.
  • Techniques for mutating antibody genes for producing aglycosylated IgG molecules are described in Tao and Morrison (1989) J. Immunol. 143:2595-2601.
  • the claimed antibody is capable of selectively binding its defined cognate antigen, i.e., the abTCR/CD3 complex.
  • the antibodies described herein bind the abTCR/CDS complex expressed on cells, including on T cells.
  • the terms“stable”,“stability”, and“stabilized”, as used herein in the context of a binding polypeptide refer to the resistance of the binding polypeptide to thermal and chemical degradation or fragmentation under given conditions of manufacture, preparation, transportation and storage.
  • The“stable” compositions retain biological activity greater than or equal to 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, or 99.9% under given manufacture, preparation, transportation and storage conditions.
  • the stability of a binding polypeptide can be assessed, for example, in terms degrees of degradation or fragmentation, or levels of particular fragments or types or sizes of aggregates, compared to a control or compared to a starting material, using methods and measurements known to those skilled in the art. Such methods and
  • AUC reduced area under the curve
  • SEC size exclusion chromatography
  • HPSEC high performance (or high pressure) size exclusion chromatography
  • LC- MS liquid chromatography-mass spectrometry
  • CGE capillary gel electrophoresis
  • SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
  • nucleic acid includes DNA molecules which encode the antibodies described herein.
  • Preferred DNA molecules which encode the antibodies described herein are expression vectors, which are suitable for
  • the terms“treat” and“treatment,” as used herein, refer to the care of a patient or subject having a disease, disorder, or condition.
  • the treatment may be directed to, but is not limited to, any one or any combination of the following: the cure of a disease, disorder, or condition; the improvement of at least one symptom of a disease, disorder, or condition; and/or a prophylactic or preventative act in which the aim is to prevent or reduce the occurrence of a disease, disorder, or condition.
  • the treatment may be directed to, but is not limited to, the cure of a disease, disorder, or condition; or the improvement of at least one symptom of a disease, disorder, or condition.
  • a subject refers to any mammal, including mice, rats, gerbils, hamsters, guinea pigs, rabbits, cats, dogs, sheep, goats, pigs, cows, horses, and primates.
  • a subject is a mammal other than a human.
  • a subject is a non-human primate.
  • a subject is a human. //. Antibodies
  • fragments of the antibodies are capable of binding the abTCR/CD3 complex. They encompass Fab, Fab', F(ab')2, and F(v) fragments, or the individual light or heavy chain variable regions or any portion thereof. Fragments include, for example, Fab, Fab', F(ab')2, Fv, scFv and the like. In certain aspects, fragments lack the Fc portion of an intact antibody, clear more rapidly from the circulation, and/or can have less non-specific tissue binding than an intact antibody. In certain aspects, fragments can be produced from intact antibodies using well known methods, for example by proteolytic cleavage with enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
  • antibodies and/or antibody fragments encompass single chain antibody fragments (scFv) that bind to the abTCR/CD3 complex.
  • an scFv comprises an antibody heavy chain variable region (VH) operabiy linked to an antibody light chain variable region (VI), wherein one or both of the heavy chain variable region and the light chain variable region, together or individually, form a binding site that binds abTCR.
  • An scFv may comprise a VH region at the amino-terminal end and a VI region at the carboxy-terminal end.
  • scFv may comprise a VL region at the amino-terminal end and a VH region at the carboxy-terminal end.
  • VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv)).
  • An scFv may optionally further comprise a polypeptide linker between the heavy chain variable region and the light chain variable region.
  • Antibodies and antibody fragments also encompass domain antibody (dAb) fragments as described in Ward, E.S. et al. (1989) Nature 341 :544-546, which consist of a VH domain.
  • Antibodies and antibody fragments also encompass heavy chain antibodies (HCAb).
  • HCAbs are reported to form antigen-binding regions using only heavy chain variable region, in that these functional antibodies are dimers of heavy chains only (referred to as“heavy-chain antibodies” or“HCAbs”). Accordingly, in certain aspects, antibodies and antibody fragments may be HCAbs that specifically bind to the abTCR/CD3 complex.
  • Antibodies and antibody fragments also encompass antibodies that are small modular immunopharmaceuticals (SMIPs) or binding domain immunoglobulin fusion proteins specific for abTCR/CD3 complex. These constructs are single-chain polypeptides comprising antigen-binding domains fused to immunoglobulin domains necessary to carry out antibody effector functions (see, WO 2005/017148). Antibodies and antibody fragments also encompass diabodies. Diabodies refer to bivalent antibodies in which VH and VI domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain. This forces the domains to pair with complementary domains of another chain and thereby creates two antigen- binding sites (see, for example, WO 93/11161 ). Diabodies can be bi-specific or mono-specific.
  • an antibody or antibody fragment specifically binds to the human abTCR/CD3 complex, i.e., such antibody or antibody fragment does not cross-react with any target other than the human abTCR/CD3 complex.
  • the antibody or antibody fragment may be modified in order to increase its serum half-life, for example, by adding molecules such as PEG or other water- soluble polymers, including polysaccharide polymers and the like to increase the half-life of the antibody or antibody fragment.
  • molecules such as PEG or other water- soluble polymers, including polysaccharide polymers and the like to increase the half-life of the antibody or antibody fragment.
  • Antibodies and antibody fragments may be multispecific or bispecific.
  • bispecific antibodies or antibody fragments may resemble single antibodies (or antibody fragments) that comprise two different binding sites (variable regions).
  • Bispecific antibodies can be produced by various methods, such as chemical techniques,“polydoma” techniques or recombinant DNA techniques.
  • Bispecific antibodies may have binding specificities for at least two different epitopes, at least one of which is the abTCR/CD3 complex. The other specificity may be selected from any useful or desired specificity including, for example, specificity for human serum albumin for the extension of half-life in vivo.
  • BLINCYTO® Philadelphia chromosome-negative relapsed or refractory acute lymphoblastic leukemia
  • ALL Philadelphia chromosome-negative relapsed or refractory acute lymphoblastic leukemia
  • These antibodies have in common a binding arm which binds to T cells and a second arm which binds to the tumor target cell, resulting in T-cell-mediated lysis of the tumor target. Also in common, these molecules recruit T cells via the CDS protein located on the cell surface.
  • An alternative to recruitment via CDS is to make use of the ab T-cell receptor ( abTCR), which is also expressed on the surface of the cell.
  • abTCR ab T-cell receptor
  • antibodies according to the present disclosure can be used to develop anti-tumor antibodies by combining a specificity for a tumor associated antigen with a specificity for the ab T-cell receptor (abTCR)
  • the human frameworks used are IGH3-23 (SEQ ID NO: 17) in the case of HEBE1 , and IGHV1 -3*01 (SEQ ID NO: 18) and IGKV3-11 *Q1 (SEQ ID NO: 19) in the case of GL1 BM.
  • binding polypeptide that specifically binds human abTCR/CD3 complex, comprising a heavy chain variable region, a light chain variable region, and a constant region, wherein:
  • the light chain variable region comprises three complementarity determining regions (CDRs) LCDR1 , LCDR2, and LCDR3 comprising the amino acid sequences set forth in SEG ID NOs: 26, 27, and 28, respectively;
  • CDRs complementarity determining regions
  • SEG ID NO: 28 comprises the amino acid sequence Q-Q-W-S-S-X 1 -X 2 -L-T, wherein X 1 is an amino acid selected from the group consisting of G, D, H, S, Y, and A, and X 2 is an amino acid selected from the group consisting of P and A; and
  • the constant region is of human origin.
  • X 1 is G.
  • X 1 is D
  • X 1 is H.
  • X 1 is S.
  • X 1 is Y.
  • X 1 is A.
  • X 2 is P. In certain embodiments, X 2 is A.
  • X 1 is Q
  • X 2 is P
  • X 1 is Q
  • X 2 is A
  • X 1 is D
  • X 2 is P
  • X 1 is D
  • X 2 is A
  • X 1 is H
  • X 2 is P
  • X 1 is H
  • X 2 is A
  • X 1 is S
  • X 2 is P
  • X 1 is S
  • X 2 is A
  • X 1 is Y
  • X 2 is P
  • X 1 is Y
  • X 2 is A
  • X 1 is A
  • X 2 is P
  • X 1 is A
  • X 2 is A
  • the light chain variable region further comprises a human light chain framework region in accordance with SEQ ID NO: 14.
  • the binding polypeptide has increased stability at pH greater than 5.0 compared to reference antibody VH31.
  • the binding polypeptide has increased stability at a temperature greater than 4°C compared to reference antibody VH31.
  • the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 7, 12, 13, 15, and 16.
  • the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 12, and SEQ ID NO: 13.
  • the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 15 and SEQ ID NO: 16.
  • the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 15.
  • the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 16.
  • Anti-abTCR antibodies may comprise one or more modifications. Modified anti-abTCR antibodies according to the invention can be made using any techniques known in the art. i) Reducing Fragmentation
  • fragmentation as used herein in the context of a binding
  • polypeptide composition refers to cleavage of the binding polypeptide or a first portion thereof into two or more portions each of lower molecular weight than the original binding polypeptide or first portion thereof.
  • fragmentation forms include, but are not limited to, a full-length unpaired heavy chain, a full-length unpaired light chain, a first full-length heavy chain paired with a second full-length heavy chain, a full-length heavy chain paired with a partial heavy chain, a first partial heavy chain paired with a second partial heavy chain, a full-length heavy chain paired with a full-length light chain, a full-length heavy chain paired with a partial light chain, a partial heavy chain paired with a full-length light chain, a partial heavy chain paired with a full-length light chain, a partial heavy chain paired with a partial light chain, a first full-length heavy chain paired with a second full-length heavy chain and a full-length light chain, a first full-length heavy chain paired with a second
  • a“fragment” refers to at least one amino acid. Typically, a fragment will comprise two or more amino acids linked together, more typically at least 10, 20, 30, 40, or 50 such amino acids. Particularly for fragments comprising more than one polypeptide chain, a fragment can comprise, for example, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, or at least 700 amino acids.
  • Fragmentation may be caused by a variety of mechanisms, e.g., due to the disruption of native covalent bonds, resulting in the cleavage of polypeptide backbones through spontaneous (e.g., non-enzymatic) or enzymatic reactions.
  • protein backbones are highly stable under physiological conditions.
  • regions and motifs may be more susceptible to fragmentation due to their amino acid sequence, the flexibility of the two- or three-dimensional polypeptide structure, and incompatible solvent and environmental conditions (e.g., temperature and pH)
  • Asn (D), Gly (G), Ser (S), Thr (T), Cys (C), and Asn (N) are particularly susceptible to cleavage in the polypeptide backbone of antibodies. See, e.g., Liu H., ef al. J. Am. Soc. Mass Spectrom. 2009; 20: 2258-2264.
  • Asn-Pro (N-P) amide bond is known to undergo complete cleavage in the presence of ammonia. See, e.g., Tarelli E. and Corran P.H. J. Peptide Res. 2003; 62: 245-251 .
  • Fragmentation may occur at any step during the manufacture or storage of biologic compositions. Because fragmentation may result in reduced potency or the presence of unwanted and potentially immunogenic species, reducing fragmentation is a key consideration in the production of any biologic therapeutic.
  • less than 5% e.g., less than 4.9, 4.8, 4.7, 4.6, 4.5,
  • the antibody is fragmented after storage for at least one month (e.g., at least two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 1 1 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, or more) at 2°C to 8°C.
  • at least one month e.g., at least two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 1 1 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, or more
  • de-immunization can be used to decrease the immunogenicity of and antibody, or antigen binding portion thereof.
  • de-immunization includes alteration of an antibody, or antigen binding portion thereof, to modify one or more T- cell epitopes (see, e.g., WO
  • VH and VI sequences from the starting antibody may be analyzed and a human T-cell epitope“map” may be generated from each V region showing the location of epitopes in relation to complementarity-determining regions (CDRs) and other key residues within the sequence.
  • Individual T-cell epitopes from the T-cell epitope map can be analyzed in order to identify alternative amino acid substitutions with a low risk of altering activity of the final antibody.
  • VH and VL sequences can be designed comprising combinations of amino acid substitutions, and these sequences can be subsequently incorporated into a range of anti-abTCR antibodies or anti-abTCR antibody fragments for use in the methods disclosed herein, which are then tested for function.
  • Complete heavy and light chain genes comprising modified V and human C regions can then be cloned into expression vectors and the subsequent plasmids can be introduced into cell lines for the production of whole antibody.
  • the antibodies can then be compared in appropriate biochemical and biological assays, and the optimal variant can be identified.
  • a humanized monoclonal antibody, or a humanized monoclonal antibody fragment may comprise an antibody constant region (e.g., an IgG constant region e.g., a human IgG constant region, e.g., a human IgG1 or lgG4 constant region) which mediates one or more effector functions.
  • an antibody constant region e.g., an IgG constant region e.g., a human IgG constant region, e.g., a human IgG1 or lgG4 constant region
  • binding of the C1 component of complement to an antibody constant region may activate the complement system.
  • Activation of complement is important in the opsonization and lysis of cell pathogens. The activation of complement also stimulates the inflammatory response and may also be involved in autoimmune hypersensitivity.
  • Fc receptors bind to receptors on various cells via the Fc region, with a Fc receptor binding site on the antibody Fc region binding to a Fc receptor (FcR) on a cell.
  • Fc receptors There are a number of Fc receptors which are specific for different classes of antibody, including IgG (gamma receptors, i.e. , Fcy receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • a humanized monoclonal antibody, or a humanized monoclonal antibody fragment binds to an Fey receptor.
  • a humanized monoclonal antibody, or a humanized monoclonal antibody fragment may comprise a constant region which is incapable of directing one or more effector functions (e.g., ADCC activity) and/or is unable to bind Fcy receptor.
  • a humanized monoclonal antibody or a humanized monoclonal antibody fragment (e.g., an anti-abTCR antibody or fragment thereof) in which at least one amino acid in one or more of the constant region domains has been deleted or otherwise altered so as to provide desired biochemical characteristics such as, e.g., reduced or enhanced effector functions, the ability to non-covalently dimerize, increased ability to localize at the site of a tumor, reduced serum half-life, and/or increased serum half-life when compared with a whole, unaltered antibody of approximately the same
  • certain antibodies, or fragments thereof, for use in the diagnostic and treatment methods described herein are domain deleted antibodies which comprise a polypeptide chain similar to an immunoglobulin heavy chain, but which lack at least a portion of one or more heavy chain domains.
  • one entire domain of the constant region of the modified antibody ill be deleted, for example, all or part of the CH2 domain will be deleted.
  • a humanized monoclonal antibody, or a humanized monoclonal antibody fragment comprises constant regions derived from different antibody isotypes (e.g., constant regions from two or more of a human IgG1 , IgG2, IgG3, or lgG4).
  • a humanized monoclonal antibody, or a humanized monoclonal antibody fragment comprises a
  • a chimeric hinge i.e., a hinge comprising hinge portions derived from hinge domains of different antibody isotypes, e.g., an upper hinge domain from an lgG4 molecule and an lgG1 middle hinge domain.
  • a humanized monoclonal antibody, or a humanized monoclonal antibody fragment e.g., an anti-abTCR antibody or fragment thereof
  • the Fc portion of a humanized monoclonal antibody, or a humanized monoclonal antibody fragment may be mutated to increase or decrease effector function using techniques known in the art.
  • the deletion or inactivation (through point mutations or other means) of a constant region domain may reduce Fc receptor binding of the circulating modified antibody thereby increasing tumor localization.
  • constant region modifications consistent with the instant disclosure moderate complement binding and thus reduce the serum half-life and nonspecific association of a conjugated cytotoxin.
  • modifications of the constant region may be used to modify disulfide linkages or oligosaccharide moieties that allow for enhanced localization due to increased antigen specificity or flexibility.
  • the resulting physiological profile, bioavaiiability and other biochemical effects of the modifications, such as tumor localization, bio- distribution and serum half-life, may easily be measured and quantified using well know immunological techniques without undue experimentation.
  • an Fc domain employed in a humanized monoclonal antibody, or a humanized monoclonal antibody fragment is an Fc variant.
  • the term“Fc variant” refers to an Fc domain having at least one amino acid substitution relative to the wild-type Fc domain from which said Fc domain is derived.
  • the Fc variant of said human lgG1 Fc domain comprises at least one amino acid substitution relative to said Fc domain.
  • amino acid substitution(s) of an Fc variant may be located at any position (i.e., any Eli convention amino acid position) within the Fc domain.
  • the Fc variant comprises a substitution at an amino acid position located in a hinge domain or portion thereof. In another embodiment, the Fc variant comprises a substitution at an amino acid position located in a CFI2 domain or portion thereof. In another embodiment, the Fc variant comprises a substitution at an amino acid position located in a CHS domain or portion thereof. In another embodiment, the Fc variant comprises a substitution at an amino acid position located in a CH4 domain or portion thereof.
  • the antibodies may employ any art-recognized Fc variant which is known to impart an improvement (e.g., reduction or enhancement) in effector function and/or FcR binding.
  • Said Fc variants may include, for example, any one of the amino acid substitutions disclosed in International PCT Publications WO88/07089A1 ,
  • a humanized monoclonal antibody, or a humanized monoclonal antibody fragment may comprise an Fc variant comprising an amino acid substitution at EU position 268 (e.g., H268D or H268E).
  • a humanized monoclonal antibody, or a humanized monoclonal antibody fragment may comprise an amino acid substitution at EU position 239 (e.g., S239D or S239E) and/or EU position 332 (e.g., I332D or I332Q).
  • a humanized monoclonal antibody, or a humanized monoclonal antibody fragment may comprise an Fc variant comprising an amino acid substitution which alters the antigen-independent effector functions of the antibody, in particular the circulating half-life of the antibody.
  • FcRn neonatal Fc receptor
  • Fc variants with improved affinity for FcRn are anticipated to have longer serum half-lives, and such molecules have useful applications in methods of treating mammals where long half-life of the administered antibody is desired, e.g., to treat a chronic disease or disorder.
  • Fc variants with decreased FcRn binding affinity are expected to have shorter half-lives, and such molecules are also useful, for example, for administration to a mammal where a shortened circulation time may be advantageous, e.g , for in vivo diagnostic imaging or in situations where the starting antibody has toxic side effects when present in the circulation for prolonged periods.
  • Fc variants with decreased FcRn binding affinity are also less likely to cross the placenta and, thus, are also useful in the treatment of diseases or disorders in pregnant women.
  • other applications in which reduced FcRn binding affinity include those applications in which localization the brain, kidney, and/or liver is desired.
  • the altered antibodies exhibit reduced transport across the epithelium of kidney glomeruli from the vasculature.
  • the altered antibodies exhibit reduced transport across the blood brain barrier (BBB) from the brain, into the vascular space.
  • BBB blood brain barrier
  • an antibody with altered FcRn binding comprises an Fc domain having one or more amino acid substitutions within the“FcRn binding loop” of an Fc domain.
  • the FcRn binding loop is comprised of amino acid residues 280- 299 (according to Eli numbering). Exemplary amino acid substitutions which altered FcRn binding activity are disclosed in International PCT Publication No.
  • a humanized monoclonal antibody, or a humanized monoclonal antibody fragment comprises an Fc domain having one or more of the following substitutions: V284E, H285E, N286D, K290E and S304D (Eli numbering).
  • a humanized monoclonal antibody, or a humanized monoclonal antibody fragment for use in the diagnostic and treatment methods described herein has a constant region, e.g., an lgG1 or lgG4 heavy chain constant region, which is altered to reduce or eliminate glycosylation.
  • an antibody may also comprise an Fc variant comprising an amino acid substitution which alters the glycosylation of the antibody.
  • said Fc variant may have reduced glycosylation (e.g., N- or C-Iinked glycosylation).
  • the Fc variant comprises reduced glycosylation of the N-linked glycan normally found at amino acid position 297 (EU numbering).
  • the antibody has an amino acid substitution near or within a glycosylation motif, for example, an N-linked glycosylation motif that contains the amino acid sequence NXT or NXS.
  • the antibody comprises an Fc variant with an amino acid substitution at amino acid position 228 or 299 (EU numbering).
  • the antibody comprises an lgG1 or lgG4 constant region comprising an S228P and a T299A mutation (EU numbering).
  • the antibodies, or fragments thereof are modified to eliminate glycosylation.
  • Such antibodies, or fragments thereof may be referred to as“agly” antibodies, or fragments thereof (e.g.,“agly” antibody fragments). While not intending to be bound by scientific theory, it is believed that agly antibodies, or fragments thereof, may have an improved safety and stability profile in vivo.
  • agly antibodies, or agly antibody fragments comprise an aglycosylated Fc region of an lgG4 antibody which is devoid of Fc ⁇ effector function thereby eliminating the potential for Fc-mediated toxicity to normal vital organs.
  • agly antibodies, or agly antibody fragments comprise an altered glycan.
  • the agly antibody or agly antibody fragment may have a reduced number of fucose residues on an N-glycan at Asn297 (N297) of the Fc region, i.e., is afucosylated.
  • the agly antibody or agly antibody fragment may have an altered number of sialic acid residues on the N-glycan at Asn297 of the Fc region.
  • a humanized monoclonal antibody, or a humanized monoclonal antibody fragment may be modified, e.g., by the covalent attachment of a molecule to the antibody such that the covalent attachment does not prevent the antibody from specifically binding to its cognate epitope.
  • a humanized monoclonal antibody may be modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, and the like. Additionally, the derivative may contain one or more non-classical amino acids.
  • a humanized monoclonal antibody, or a humanized monoclonal antibody fragment may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions.
  • anti-abTCR antibodies may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495, WO 91/14438, and WO 89/12824; U.S. Pat. No. 5,314,995; and EP 398,387
  • a humanized monoclonal antibody, or a humanized monoclonal antibody fragment may be fused to one or more heterologous polypeptides to increase the in vivo half-life or for use in immunoassays using methods known in the art.
  • polyethylene glycol (PEG) can be conjugated to a humanized monoclonal antibody, or a humanized monoclonal antibody fragment (e.g., an anti-abTCR antibody or fragment thereof) to increase its half-life in vivo.
  • PEG polyethylene glycol
  • a humanized monoclonal antibody, or a humanized monoclonal antibody fragment can be fused to one or more marker sequences, such as a peptide, to facilitate purification or detection of the humanized monoclonal antibody, or humanized monoclonal antibody fragment (e.g., an anti-abTCR antibody or fragment thereof).
  • marker sequences such as a peptide
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311 ), among others, many of which are commercially available.
  • a pQE vector QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311
  • hexa-histidine provides for convenient purification of the fusion protein.
  • peptide tags useful for purification include, but are not limited to, the“HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767-778 (1984)) and the“flag” tag.
  • a humanized monoclonal antibody, or a humanized monoclonal antibody fragment may be used in non- conjugated form or may be conjugated to at least one of a variety of molecules, e.g., to improve the therapeutic properties of the molecule, to facilitate target detection, or for imaging or therapy of the subject
  • a humanized monoclonal antibody, or a humanized monoclonal antibody fragment e.g., an anti-abTCR antibody or fragment thereof
  • a humanized monoclonal antibody, or a humanized monoclonal antibody fragment may be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG
  • the present disclosure further encompasses a humanized monoclonal antibody, or a humanized monoclonal antibody fragment (e.g., an anti-abTCR antibody or fragment thereof) conjugated to a diagnostic or therapeutic agent.
  • a humanized monoclonal antibody, or a humanized monoclonal antibody fragment e.g., an anti-asbTCR antibody or fragment thereof
  • Detection can be facilitated by coupling a humanized monoclonal antibody, or a humanized monoclonal antibody fragment (e.g., an anti-abTCR antibody or fragment thereof) to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. See, for example, U.S. Pat. No.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, b-galactosidase, and acetylcholinesterase
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
  • D humanized monoclonal antibody, or a humanized monoclonal antibody fragment for use in the diagnostic and treatment methods disclosed herein may be conjugated to one or more cytotoxins (such as radioisotopes, cytotoxic drugs, or toxins) therapeutic agents, cytostatic agents, biological toxins, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, immunologically active ligands (e.g., lymphokines or other antibodies wherein the resulting molecule binds to both the neoplastic cell and an effector cell such as a T cell), or PEG.
  • cytotoxins such as radioisotopes, cytotoxic drugs, or toxins
  • a humanized monoclonal antibody, or a humanized monoclonal antibody fragment for use in the diagnostic and treatment methods disclosed herein can be conjugated to a molecule that decreases tumor cell growth.
  • the disclosed compositions may comprise a humanized monoclonal antibody, or a humanized monoclonal antibody fragment (e.g., an anti-abTCR antibody or fragment thereof) coupled to drugs or prodrugs.
  • Still other embodiments described herein comprise the use of antibodies, or fragments thereof, conjugated to specific biotoxins or their cytotoxic fragments such as ricin, gelonin, Pseudomonas exotoxin or diphtheria toxin or the like.
  • cytotoxic fragments such as ricin, gelonin, Pseudomonas exotoxin or diphtheria toxin or the like.
  • radioisotopes include, but are not limited to: 67 Cu, 67 Ga, 30 Y, 105 Rh, 11 1 ln, 123 l, 125 l, 131 l, 153 Sm, 166 Ho, 177 Lu, 186 Re, and 188 Re.
  • the radionuclides act by producing ionizing radiation which causes multiple strand breaks in nuclear DNA, leading to cell death.
  • the isotopes used to produce therapeutic conjugates typically produce high energy alpha- or beta-particles which have a short path length. Such radionuclides kill cells to which they are in close proximity, for example neoplastic cells to which the conjugate has attached or has entered. They have little or no effect on non-localized cells. Radionuclides are essentially non-immunogenic.
  • Antibody production can be performed by any technique known in the art, including in transgenic organisms such as goats (see, Pollock et al. (1999) J.
  • Antibodies may also be produced by chemical synthesis or by expression of genes encoding the antibodies in host cells.
  • a polynucleotide encoding a humanized monoclonal antibody, or a humanized monoclonal antibody fragment is isolated and inserted into a replicable construct or vector such as a plasmid for further propagation or expression in a host cell.
  • Constructs or vectors e.g. , expression vectors suitable for the expression of a humanized monoclonal antibody, or a humanized monoclonal antibody fragment (e.g. , an anti-abTCR antibody or fragment thereof) according to the described embodiments are available in the art.
  • vectors are available, including vectors which are maintained in single copy or multiple copies in a host cell, or which become integrated into the host cell’s chromosome(s).
  • the constructs or vectors can be introduced into a suitable host cell, and cells which express a humanized immunoglobulin can be produced and maintained in culture.
  • a single vector or multiple vectors can be used for the expression of a humanized immunoglobulin.
  • Polynucleotides encoding a humanized monoclonal antibody, or a humanized monoclonal antibody fragment are readily isolated and sequenced using conventional procedures (e.g.,
  • Vectors that may be used include plasmid, virus, phage, transposons, minichromosomes of which plasmids are a typical embodiment.
  • such vectors further include a signal sequence, origin of replication, one or more marker genes, an enhancer element, a promoter and transcription
  • polynucleotides encoding the light and heavy chains may be inserted into separate vectors and introduced (e.g., by transformation, transfection, electroporation or transduction) into the same host cell concurrently or sequentially or, if desired, both the heavy chain and light chain can be inserted into the same vector prior to such introduction.
  • a promoter can be provided for expression in a suitable host cell. Promoters can be constitutive or inducible. For example, a promoter can be operably linked to a nucleic acid encoding a humanized immunoglobulin or immunoglobulin chain, such that it directs expression of the encoded polypeptide.
  • suitable promoters for prokaryotic and eukaryotic hosts are available. Prokaryotic promoters include lac, tac, T3, T7 promoters for E.
  • Eukaryotic promoters include inducible yeast promoters such as alcohol dehydrogenase 2, isocytochrome C, acid
  • RNA polymerase II promoters including viral promoters such as polyoma, fowlpox and adenoviruses (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus (in particular, the immediate early gene promoter), retrovirus, hepatitis B virus, actin, Rous sarcoma virus (RSV) promoter and the early or late Simian virus 40 and non-viral promoters such as EF-1 alpha (Mizushima and Nagata (1990) Nucleic Acids Res. 18(17):5322).
  • viral promoters such as polyoma, fowlpox and adenoviruses (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus (in particular, the immediate early gene promoter), retrovirus, hepatitis B virus
  • enhancer elements can be included instead of or as well as those found located in the promoters described above.
  • Suitable mammalian enhancer sequences include enhancer elements from giobin, elastase, albumin, fetoprotein,
  • an enhancer element from a eukaryotic cell virus such as SV40 enhancer, cytomegalovirus early promoter enhancer, polyoma enhancer, baculoviral enhancer or murine lgG2a locus (see, WO 04/09823). Whilst such enhancers are often located on the vector at a site upstream to the promoter, they can also be located elsewhere e.g., within the untranslated region or downstream of the polyadenylation signal. The choice and positioning of enhancer may be based upon compatibility with the host cell used for expression.
  • the vectors may comprise a selectable marker for selection of host cells carrying the vector, and, in the case of a replicable vector, an origin of replication.
  • Genes encoding products which confer antibiotic or drug resistance are common selectable markers and may be used in prokaryotic (e.g., f3-lactamase gene (ampicillin resistance), tet gene (tetracycline resistance) and eukaryotic cells (e.g., neomycin (G418 or geneticin), gpt (mycophenolic acid), ampiciilin, or bygromycin 5 resistance genes).
  • Dihydrofolate reductase marker genes permit selection with methotrexate in a variety of hosts.
  • auxotrophic markers of the host e.g., LEU2, URA3, HISS
  • viral e.g., baculovirus
  • phage vectors and vectors which are capable of integrating into the genome of the host cell, such as retroviral vectors, are also contemplated.
  • polyadenylation and termination signals are operabiy linked to polynucleotide encoding the antibody described herein. Such signals are typically placed 3' of the open reading frame.
  • polyadenylation/termination signals include those derived from growth hormones, elongation factor-1 alpha and viral (e.g., SV40) genes or retroviral long terminal repeats.
  • elongation factor-1 alpha elongation factor-1 alpha
  • viral e.g., SV40
  • polyadenylation/termination signals include those derived from the phosphoglycerate kinase (PGK) and the alcohol dehydrogenase 1 ( ADH ) genes.
  • PGK phosphoglycerate kinase
  • ADH alcohol dehydrogenase 1
  • polyadenylation/termination sequences may be based upon compatibility with the host cell used for expression.
  • other features that can be employed to enhance yields include chromatin remodeling elements, introns and host cell specific codon modification.
  • the codon usage of the antibodies described herein can be modified to accommodate codon bias of the host cell such to augment transcript and/or product yield (e.g., Hoekema, A. et al. (1987) Mol. Cell Biol.
  • codons may be based upon compatibility with the host cell used for expression.
  • This disclosure thus relates to isolated nucleic acid molecules that encode the humanized immunoglobulins, or heavy or light chains, thereof. This disclosure also relates to isolated nucleic acid molecules that encode an antigen-binding portion of the immunoglobulins and their chains.
  • a humanized monoclonal antibody, or a humanized monoclonal antibody fragment can be produced, for example, by the expression of one or more recombinant nucleic acids encoding the antibody in a suitable host cell.
  • the host cell can be produced using any suitable method.
  • the expression constructs e.g., one or more vectors, e.g., a mammalian cell expression vector
  • the resulting cell can be maintained (e.g., in culture, in an animal, in a plant) under conditions suitable for expression of the construct(s) or vector(s).
  • Host cells can be prokaryotic, including bacterial cells such as E. coli (e.g., strain DH5a TM ) (Invitrogen, Carlsbad, CA), PerC6 (Crucell, Leiden, NL), B. subfilis and/or other suitable bacteria; eukaryotic cells, such as fungal or yeast cells (e.g., Pichia pastoris, Aspergillus sp.
  • E. coli e.g., strain DH5a TM
  • PerC6 Cell, Leiden, NL
  • B. subfilis and/or other suitable bacteria
  • eukaryotic cells such as fungal or yeast cells (e.g., Pichia pastoris, Aspergillus sp.
  • insects e.g., Drosophila Schnieder S2 cells, Sf9 insect cells
  • BTI-TN-5B1-4 High FiveTM insect cells
  • mammals e.g., COS cells, such as COS-1 (ATCC Accession No. CRL-1650) and COS-7 (ATCC Accession No. CRL-1651 )
  • CHO e.g., ATCC Accession No. CRL- 9096
  • CHO DG44 Urlaub, G. and Chasin, L.A. (1980) Proc. Natl. Acad. Sci. USA, 77(7):4216-4220
  • 293 ATCC Accession No. CRL-1573
  • HEK HeLa
  • the host cell is not part of a multicellular organism (e.g., plant or animal), e.g , it is an isolated host cell or is part of a cell culture.
  • Host cells may be cultured in spinner flasks, shake flasks, roller bottles, wave reactors (e.g., System 1000 from wavebiotech.com) or hollow fiber systems, but it is preferred for large scale production that stirred tank reactors or bag reactors (e.g., Wave Biotech, Somerset, New Jersey USA) are used particularly for suspension cultures.
  • Stirred tank reactors can be adapted for aeration using e.g., spargers, baffles or low shear impellers. For bubble columns and airlift reactors, direct aeration with air or oxygen bubbles maybe used.
  • the medium can be supplemented with a cell protective agent such as pluronic F-68 to help prevent cell damage as a result of the aeration process.
  • a cell protective agent such as pluronic F-68 to help prevent cell damage as a result of the aeration process.
  • microcarriers may be used as growth substrates for anchorage dependent cell lines, or the cells may be adapted to suspension culture.
  • the culturing of host cells, particularly vertebrate host cells may utilize a variety of operational modes such as batch, fed-batch, repeated batch processing (see, Drapeau et al. (1994) Cytotechnology 15:103-109), extended batch process or perfusion culture.
  • recombinantly transformed mammalian host cells may be cultured in serum-containing media such media comprising fetal calf serum (FCS), it is preferred that such host cells are cultured in serum-free media such as disclosed in Keen et al. (1995) Cytotechnology 17:153-163, or commercially available media such as ProCHOTM or UltraCHOTM (Cambrex NJ, USA),
  • the serum-free culturing of host cells may require that those cells are adapted to grow in serum-free conditions.
  • One adaptation approach is to culture such host cells in serum containing media and repeatedly exchange 80% of the culture medium for the serum-free media so that the host cells learn to adapt in serum-free conditions (see, e.g., Scharfenberg, K. et al. (1995) Animal Cell Technology: Developments Towards the 21 st Century
  • a humanized monoclonal antibody, or a humanized monoclonal antibody fragment may be secreted into the medium and recovered and purified therefrom using a variety of techniques to provide a degree of purification suitable for the intended use.
  • a humanized monoclonal antibody, or a humanized monoclonal antibody fragment e.g., an anti-abTCR antibody or fragment thereof
  • the use of a humanized monoclonal antibody, or a humanized monoclonal antibody fragment (e.g., an anti-abTCR antibody or fragment thereof) for the treatment of human subjects typically mandates at least 95% purity as determined by reducing SDS-PAGE, more typically 98% or 99% purity, when compared to the culture media comprising the therapeutic antibodies.
  • cell debris from the culture media can be removed using centrifugation followed by a clarification step of the supernatant using e.g., microfiltration, ultrafiltration and/or depth filtration.
  • a humanized monoclonal antibody, or a humanized monoclonal antibody fragment e.g., an anti- abTCR antibody or fragment thereof
  • microfiltration e.g., a cell debris from the culture media
  • a humanized monoclonal antibody, or a humanized monoclonal antibody fragment e.g., an anti- abTCR antibody or fragment thereof
  • a humanized monoclonal antibody, or a humanized monoclonal antibody fragment (e.g., an anti-abTCR antibody or fragment thereof), following various clarification steps, are captured using Protein A or Protein G affinity chromatography followed by further chromatography steps such as ion exchange and/or HA chromatography, anion or cation exchange, size exclusion chromatography and ammonium sulphate precipitation.
  • Various virus removal steps may also be employed (e.g., nanofiltration using, e.g., a DV-20 filter).
  • a purified preparation comprising at least 10 mg/mL or greater, e.g., 100 mg/mL or greater of the antibody described herein is provided and, therefore, forms another embodiment described herein. Concentration to 100 mg/mL or greater can be generated by
  • Bacterial systems are particularly suited for the expression of antibody fragments. Such fragments are localized intracellularly or within the periplasm.
  • Insoluble periplasmic proteins can be extracted and refolded to form active proteins according to methods known to those skilled in the art, see, Sanchez et al. (1999) J. Biotechnol. 72:13-20; Cupit, P.M. et al. (1999) Lett. Appl. Microbiol. 29:273-277.
  • the present disclosure also relates to cells comprising a nucleic acid, e.g., a vector, described herein (e.g., an expression vector).
  • a nucleic acid i.e., one or more nucleic acids
  • a construct e.g., one or more constructs, e.g , one or more vectors
  • nucleic acid(s) can be introduced into a suitable host cell by a method appropriate to the host cell selected (e.g., transformation, transfection, electroporation, infection), with the nucleic acid(s) being, or becoming, operabiy linked to one or more expression control elements (e.g., in a vector, in a construct created by processes in the cell, integrated into the host cell genome).
  • Host cells can be maintained under conditions suitable for expression (e.g., in the presence of inducer, suitable media supplemented with appropriate salts, growth factors, antibiotic, nutritional supplements, etc.), whereby the encoded polypeptide(s) are produced.
  • the encoded humanized antibody can be isolated, for example, from the host cells, culture medium, or milk. This process encompasses expression in a host cell (e.g., a mammary gland cell) of a transgenic animal or plant (e.g., tobacco) (see, e.g., WO 92/03918).
  • T-cell activity is desirable in a number of situations in which immunosuppression is warranted, and/or an autoimmune condition occurs.
  • targeting of the abTCR/CD3 complex is indicated in the treatment of diseases involving an inappropriate or undesired immune response, such as inflammation, autoimmunity, and/or other conditions involving such mechanisms.
  • diseases involving an inappropriate or undesired immune response such as inflammation, autoimmunity, and/or other conditions involving such mechanisms.
  • disease or disorder is an autoimmune and/or inflammatory disease.
  • T-cell mediated diseases include but are not limited to: systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), inflammatory bowel disease (IBD) (including ulcerative colitis (UC) and Crohn’s disease (CD)), multiple sclerosis (MS), scleroderma, type 1 diabetes (T1 D), and other diseases and disorders, such as pemphigus vulgaris (PV), psoriasis, atopic dermatitis, celiac disease, chronic obstructive lung disease,
  • SLE systemic lupus erythematosus
  • RA rheumatoid arthritis
  • IBD inflammatory bowel disease
  • MS multiple sclerosis
  • T1 D type 1 diabetes
  • diseases and disorders such as pemphigus vulgaris (PV), psoriasis, atopic dermatitis, celiac disease, chronic obstructive lung disease
  • Hashimoto s thyroiditis, Graves’ disease (thyroid), Sjogren ' s syndrome, Guillain- Barre syndrome, Goodpasture’s syndrome, Addison’s disease, Wegener’s
  • granulomatosis primary biliary sclerosis, sclerosing cholangitis, autoimmune hepatitis, polymyalgia rheumatica, Raynaud’s phenomenon, temporal arteritis, giant cell arteritis, autoimmune hemolytic anemia, pernicious anemia, polyarteritis nodosa, Behcet’s disease, primary biliary cirrhosis, uveitis, myocarditis, rheumatic fever, ankylosing spondylitis, glomerulonephritis, sarcoidosis, dermatomyositis, myasthenia gravis, polymyositis, alopecia areata, and vitiigo.
  • such disease or disorder is SLE. In one embodiment, such disease or disorder is RA. In one embodiment, such disease or disorder is IBD. In one embodiment, such disease or disorder is MS In one embodiment, such disease or disorder is T1 D.
  • such disease or disorder is a xenotransplant, an allotransplant, a xenopregnancy, pre-eclampsia, or Rh disease.
  • the antibodies according to the described antibodies are identical to the antibodies according to the described antibodies.
  • embodiments are used to aid transplantation by immunosuppressing the subject.
  • Such use alleviates graft-versus-host disease (GvHD), a common complication following xenograft or allograft (including, but not limited to: the transplant of stem cells, bone marrow, tissues, body parts, and solid organs).
  • Tissues may include, but are not limited to: cornea, sclera, bone, skin, blood vessels, and heart valves.
  • Body parts may include, but are not limited to: a face or a portion thereof, one or more arms or portions thereof, one or more hands or portions thereof, one or more legs or portions thereof, and a scalp or portions thereof.
  • Solid organs may include, but are not limited to: heart, lung, liver, kidney, pancreas, stomach, small intestine, large intestine, testis and ovary.
  • existing treatments for graft-versus- host disease see, e.g , Svennilson, Bone Marrow Transplantation (2005) 35:885- S67, and references cited therein.
  • the antibodies presented in this disclosure may be used in combination with other available therapies.
  • combination therapy may include administration of an antibody described herein together with a medicament, which together with the antibody comprises an effective amount for preventing or treating such autoimmune diseases.
  • the combination therapy may encompass one or more of an agent that promotes the growth of pancreatic beta-cells or enhances beta-cell transplantation, such as beta cell growth or survival factors or immunomodulatory antibodies.
  • said combination therapy may encompass one or more of methotrexate, an anti-TNF-a antibody, a TNF-a receptor-lg fusion protein, an anti-IL-15 or anti-IL-21 antibody, a non-steroidal anti-inflammatory drug (NSAID), or a disease-modifying anti-rheumatic drug
  • the additional agent may be a biological agent such as an anti-TNF agent (e.g., ENBREL®, infliximab (REMICADE®) and adalimumab
  • an anti-TNF agent e.g., ENBREL®, infliximab (REMICADE®) and adalimumab
  • hematopoietic growth factor(s) such as erythropoietin, G-CSF, GM-CSF, IL-3, !L-11 , thrombopoietin, etc.
  • antimicrobial(s) such as antibacterial, antiviral, antifungal drugs
  • the additional agent may be one or more of tar and derivatives thereof, phototherapy, corticosteroids, cyclosporine A, vitamin D analogs, methotrexate, p38 mitogen-activated protein kinase (MARK) inhibitors, as well as biologic agents such as anti-TNF-a agents and RITUXAN®.
  • said autoimmune disease is an inflammatory bowel disease (IBD) such as, for example, Crohn’s disease or ulcerative colitis
  • IBD inflammatory bowel disease
  • the additional agent may be one or more of
  • aminosalicylates corticosteroids, immunomodulators, antibiotics, or biologic agents such as REMICADE® and HUMIRA®.
  • the combination treatment may be carried out in any way as deemed necessary or convenient by the person skilled in the art and for the purpose of this specification, no limitations with regard to the order, amount, repetition or relative amount of the compounds to be used in combination is contemplated. Accordingly, the antibodies according to the described embodiments may be formulated info pharmaceutical compositions for use in therapy.
  • compositions comprising a
  • Ligands may be immunoglobulins, peptides, nucleic acids or small molecules, as discussed herein. They are referred to, in the following discussion, as“compounds.”
  • a pharmaceutical composition described herein is a composition of matter comprising a compound or compounds capable of modulating T-cell activity as an active ingredient.
  • the compound is in the form of any pharmaceutically acceptable salt, or e.g., where appropriate, is an analog, free base form, tautomer, enantiomer racemate, or combination thereof.
  • the active ingredients of a pharmaceutical composition comprising the active ingredient described herein are contemplated to exhibit therapeutic activity, for example, in the treatment of graft-versus-host disease, when administered in an amount which depends on the particular case.
  • a pharmaceutical composition comprises a
  • humanized monoclonal antibody or a humanized monoclonal antibody fragment (e.g., an anti-asbTCR antibody or fragment thereof) described herein, and
  • one or more compounds described in this disclosure may be used in combination with any art recognized compound known to be suitable for treating the particular indication in treating any of the aforementioned conditions. Accordingly, one or more compounds described herein may be combined with one or more art recognized compounds known to be suitable for treating the foregoing indications such that a convenient, single composition can be administered to the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response.
  • doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • the active ingredient may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intramuscular, subcutaneous, intranasal, intradermal or suppository routes or implanting (e.g., using slow release molecules).
  • the active ingredient may also be used to treat cells, tissues, or organs being transplanted into a patient prior to the transplantation. This may be done in order to prevent, decrease the likelihood, or lessen the symptoms of, for example, graft versus host disease.
  • the active ingredient may be required to be coated in a material to protect said ingredients from the action of enzymes, acids and other natural conditions which may inactivate said ingredient.
  • the active ingredient in order to administer the active ingredient by means other than parenteral administration, it will be coated by, or administered with, a material to prevent its inactivation.
  • the active ingredient may be administered in an adjuvant, co-administered with enzyme inhibitors or in liposomes.
  • Adjuvant is used in its broadest sense and includes any immune stimulating compound such as interferon.
  • Adjuvants contemplated herein include resorcinols, non-ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether.
  • Enzyme inhibitors include pancreatic trypsin.
  • Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes.
  • the active ingredient may also be administered parenterally or
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid
  • polyethylene glycol, and the like suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active ingredient in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-fiitered solution thereof.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active ingredient may be incorporated into sustained-release preparations and formulations.
  • pharmaceutically acceptable carrier and/or diluent includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the pharmaceutically acceptable carrier or diluent is an aqueous fluid.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated.
  • Supplementary active ingredients can also be incorporated into the compositions.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the novel dosage unit forms described herein are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such as active material for the treatment of disease in living subjects having a disease or condition in which bodily health is impaired.
  • the principal active ingredients are compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
  • Dosages may include, but are not limited to, 0.01 mg/kg to 20 mg/kg, including: 0 01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0 04 mg/kg, 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg, 0.08 mg/kg, 0.09 mg/kg, 0.10 mg/kg, , 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 .0 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1 .3 mg/kg, 1 .4 mg/kg, 1 .5 mg/kg, 1 .6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1 .9 mg/kg, 2.0 mg/kg, 2.1 mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5 mg/kg, 2.6 mg/kg, 2.7 mg/kg
  • Dosages may also include, but are not limited to: about 100 ng/kg to about 0.01 mg/kg, including: about 100 ng/kg, about 200 ng/kg, about 300 ng/kg, about 400 ng/kg, about 500 ng/kg, about 600 ng/kg, about 700 ng/kg, about 800 ng/kg, about 900 ng/kg, about 1 microgram/kg, about 2 microgram/kg, about 3 microgram/kg, about 4 microgram/kg, about 5 microgram/kg, about 6 microgram/kg, about 7 microgram/kg, about 8 microgram/kg, about 9 microgram/kg, about 10 microgram/kg.
  • peptides may be modified in order to improve their ability to cross a cell membrane.
  • US 5,149,782 discloses the use of fusogenic peptides, ion- channel forming peptides, membrane peptides, long-chain fatty acids and other membrane blending agents to increase protein transport across the cell membrane.
  • a method for treating a condition associated with an aberrant immune response comprising administering to a subject a
  • the reference or control VH31 antibody is a wild-type humanized anti-human abTCR antibody in the GL1 BM series, comprising a heavy chain variable domain with the amino acid sequence set forth as SEG ID NO: 16 and a light chain variable domain with the amino acid sequence set forth as SEQ ID NO: 14 (Table 1 ). No fragmentation was observed in liquid formulations after four months at
  • the VH31 anti-abTCR antibody selectively depletes activated T cells.
  • a cell-based potency assay was performed. The potency assay is based on the activation of the T-cell receptor and the subsequent binding of the anti- abTCR antibody to stimulate the apoptotic pathway.
  • Jurkat T lymphocytes express the T-cell receptor complex.
  • the potency assay utilizes a Jurkat T-cell line containing a nuclear factor of activated T cells (NFAT) response element linked to a luciferase reporter. This engineered cell line is used to examine T-cell activation and subsequent inhibition of the activation signal by the anti-asbTCR antibody. Activation of the T-cell receptor complex stimulates the NFAT pathway leading to Iuciferase production. Luciferase production is then measured using luciferase substrate (e.g., luciferin) and a luminescence detector. The level of activation is proportional to luciferase production.
  • luciferase substrate e.g., luciferin
  • Immobilized anti-CD3 activates the Jurkat T cells, as measured through induction of the NFAT pathway and subsequent luciferase production in the NFAT-luciferase Jurkat cells. Activation is inhibited in a dose dependent manner by the anti-abTCR antibody, which is a qualitative measure of anti-abTCR potency.
  • the qualitative potency assay was run in a 96-well plate format.
  • the 96-well plates were coated with 1 mg/mL of anti-CD3 at 2-8°C overnight.
  • the plates were washed and NFAT-luciferase Jurkat T lymphocytes (5 x 10 4 cells/well) were added to the anti-CD3-coated plates.
  • the cells were incubated with anti ⁇ CD3 at 37°C and 5% CO2 for five hours, in order to activate the T-cell receptor complex.
  • Each cell condition was then incubated with multiple levels (0.003 to 200 mg/mL) of anti- abTCR (control and sample) for an additional eighteen hours.
  • Negative anti-CD3 (0 mg/mL) and anti-abTCR (0 pg/ml) controls were included for each condition.
  • the anti-abTCR dilutions were removed and the cells were lysed with cell culture lysis reagent.
  • the lysates were measured for luciferase production using luciferase substrate and a luminescence plate reader. Higher doses of anti-abTCR antibody are expected to result in a lower luminescent signal, indicating inhibition of the activation response.
  • Dose response data were plotted using Softmax Pro software and sample dose response curves were visually compared to control dose response curves.
  • the graph in FIG. 2 shows data from the cell-based potency assay.
  • the VH31 antibody formulation exposed to accelerated conditions (i.e., 45°G; pH 8 0) for five weeks (open square symbols) showed considerable loss of potency compared to the VH31 formulation exposed to less severe conditions (i.e , 45°C; pH 5.0) for five weeks (open circle symbols) or the control conditions (i.e., ⁇ 0°C; pH 5.0; open diamond symbols).
  • This study demonstrated a relationship between the appearance of the LMW species and a loss of antibody potency, likely due to light chain fragmentation.
  • Antibody fragments were further analyzed by molecular weight estimation of the observed bands, as well as through N-terminal sequencing and peptide mapping studies (FIG. 3). Chemical and enzymatic cleavage of polypeptides is known to occur between Asn (N) and Pro (P) residues. Therefore, based on sequencing and mapping studies, light chain amino acid residues N93 and P94 were identified as the probable location of light chain clipping and the source of the identified fragments (FIG. 4). To further confirm this, the VH31 light chain (P7) was subjected to trypsin digestion at pH 4.0 to 8.0. The graphs in FIG. 5 show the various light chain fragments that were expected if enzymatic clipping were to occur at N93/P94.
  • the amino acid Pro (P) provides little freedom in folded protein structures. Therefore, it was postulated that it may be difficult to replace Pro at residue 94 without destabilizing the conformation of the LC CDR3.
  • Ala (A) is commonly used in mutagenesis due to its physical characteristics. As a starting point, residues N93 and P94 were either individually or concomitantly replaced with A. In addition, several other substitutions were made at residue N93, based on amino acid characteristics that were thought to be compatible, e.g., charge, size, H-bonding, polar vs. non- polar, etc. FIG. 7 shows the various amino acid substitutions that were made and tested.
  • Wild-type anti-abTCR VH31 expression vector was used as the template for mutagenesis and expression control. Eight pairs of primers were designed, and PCR mutagenesis was performed using the GuikChange Lightning Site-Directed
  • Mutant and wild-type (WT) DNA were transfected into the Expi293 transient expression system.
  • Conditioned media were harvested four days post-transfection, and expression levels were measured by Octet Protein A assay.
  • the results shown in Table 2) indicated good expression for the abTCR wild-type (114 mg/mL media) and 7 of the 8 mutants (>45 mg/mL media).
  • One mutant (N93A) was expressed at a relatively lower level (16 mg/mL) The transfection was scaled up to 30 mL for all 8 mutants.
  • Conditioned media were collected for Protein A purification.
  • the conditioned media were put through the 1 mL HiTrap Protein A HP (GE) column for purification.
  • the column was equilibrated with PBS pH 7.2 (Gibco) for 5 column volumes.
  • the media were loaded at a 0.5 mL/min flow rate and then washed with PBS pH 7.2 for 20 column volumes (CVs) before elution.
  • Antibody was eluted into 10 mM succinate, pH 3.75 and adjusted to pH 5.5 with 0.2 M sodium hydroxide right after elution. All samples were filtered with 0.2 mm low-protein binding membrane with a syringe filter (recovery 50%-80%) and Amicon YM30 was used to concentrate the sample (recovery 100%).
  • Table 2 The results are summarized in Table 2.
  • HiTrap Protein A HP (GE) column (1 mL) was used for purification. The column was equilibrated with PBS pH 7.2 (Gibco) for 5 CVs. Conditioned media sample was loaded at 0.5 mL/min flow rate and then washed with PBS pH 7.2 for 20 CVs before elution. Antibody was eluted into 10 mM succinate, pH 3.75 for 5 CVs (5 mL in total) and the yield was almost 100%. Eluted antibody was adjusted to pH 5.5 with 0.2 IV! sodium hydroxide after elution. Solutions turned slightly cloudy at pH 5.5 and were filtered with 50 mL tube top filters (0.2 pm CA, low protein binding).
  • Example 2 The qualitative potency assay described in Example 2 was repeated with antibodies comprising the variant iight chains.
  • the wildtype and controi antibodies comprised the identical VH31 amino acid sequence, but were produced from different preparations.
  • the wildtype VH31 antibody was produced together with the LC variant antibodies, whereas the control antibody was produced from a separate large-scale preparation.
  • an antibody comprising LC variant N93S was only slightly less potent than either the wildtype VH31 or controi antibody.
  • antibodies comprising LC variants N93Q, N93A, and N93A/P94A were ail markedly less potent than any of the aforementioned antibodies. Based on these results, the N93S variant was selected as a candidate for accelerated stability studies.
  • Example 7 Accelerated Stability Studies of LC Variants
  • the antibody comprising the LC variant N93S was subjected to increased temperature (i.e. , 45°C) and pH (i.e., pH 8.0) for six weeks to determine whether the N93S amino acid substitution stabilized the LC and prevented fragmentation.
  • pH i.e., pH 8.0
  • fragmentation was observed in wildtype, reference VH31 , and N93S antibodies.
  • the N93S mutant had demonstrably reduced degradation compared to either the wiidtype or VH31 control antibodies (FIG. 11 ).
  • the N93S mutant had markedly reduced loss of potency under accelerated conditions (3-fold loss) compared to either the wildtype or VH31 control antibodies (each greater than 20-fold loss).
  • pH 5.5 which is closer to the pH of a hypothetical antibody drug substance formulation than pH 8.0, fragmentation was noticeably less prevalent at six weeks (FIG. 12)

Abstract

La présente invention concerne des compositions et des procédés améliorés permettant de traiter des maladies et des troubles à médiation par des lymphocytes T (par exemple, des troubles auto-immuns, une maladie du greffon contre l'hôte et un rejet de greffe). L'invention concerne des polypeptides de liaison anti-apβTCR, comprenant des anticorps, qui comprennent au moins une substitution ou une modification d'acide aminé qui augmente la stabilité du polypeptide de liaison par réduction de la fragmentation de la région variable de chaîne légère. Les procédés selon l'invention impliquent généralement l'administration à un sujet qui en a besoin d'une quantité efficace d'un polypeptide de liaison humanisé stabilisé qui est spécifique du récepteur des lymphocytes T bêta (apβTCR).
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US20220153840A1 (en) 2022-05-19
CN113939534A (zh) 2022-01-14
CA3135032A1 (fr) 2020-10-08
AU2020253455A1 (en) 2021-11-04
WO2020206063A1 (fr) 2020-10-08
MA55529A (fr) 2022-02-09
JP2022521850A (ja) 2022-04-12

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